Human sphingosine-1-phosphate phosphatase and inhibition methods

ABSTRACT

The present invention provides polynucleotides and polypeptides of a human sphingosine-1-phosphate phosphatase, referred to herein as hSPP1. The polynucleotides and polypeptides are used to further provide expression vectors, host cells comprising the vectors, probes and primers, antibodies against the hSPP1 protein and polypeptides thereof, assays for the presence or expression of hSPP1 and assays for the identification of compounds that interact with hSPP1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/US02/03833 filed Feb. 6, 2002 whichclaims benefit to U.S. Provisional Application No. 60/267,000 filed onFeb. 7, 2001.

FIELD OF THE INVENTION

The invention relates to human sphingosine-1-phosphate phosphatase,polynucleotides encoding the enzyme and assays that measure thecatabolism of sphingosine-1-phosphate by human sphingosine-1-phosphatephosphatase.

BACKGROUND OF THE INVENTION

Sphingosine-1-phosphate (SPP) is a bioactive sphingolipid metabolitewhich regulates diverse biological processes (reviewed in (Goetzl, etal., (1998) FASEB J.12, 1589-1598 and Spiegel, S. (1999) J. Leukoc.Biol. 65, 341-344.) Many of its actions are reported to be mediated by afamily of specific cell surface G-protein coupled receptors (GPCR),known as EDG (endothelial differentiation genes) receptors. Binding ofSPP to EDG-1 expressed on endothelial cells reportedly enhances survival(Hisano, et al., (1999) Blood 93, 4293-4299), chemotaxis and in vitroangiogenesis (Wang, et al., (1999) J. Biol. Chem. 274, 35343-35350) andadherens junction assembly leading to morphogenetic differentiation(Lee, et al., (1999) Cell 99, 301-312), whereas binding of SPP to EDG-5and EDG-3 is reported to induce neurite retraction and soma rounding(Postma, et al., (1996) EMBO J. 15, 2388-2392 and Van Brocklyn, et al.,(1999) J. Biol. Chem. 274, 46264632). Additional research indicates thatSPP induces activation of G_(i)-gated inward rectifying K+-channels inatrial myocytes (van Koppen, et al., (1996) J. Biol. Chem. 271,2082-2087) and inhibits motility of melanoma cells (Yamamura, et al.,(1997) Biochemistry 36, 10751-10759) through as yet uncharacterizedGPCRs.

SPP is also described as performing important roles inside cells. Inresponse to diverse external stimuli, sphingosine kinase, the enzymethat catalyzes the phosphorylation of sphingosine to SPP, is activated(Olivera, et al., (1993) Nature 365, 557-560; Choi, et al., (1996)Nature 380, 634-636; Melendez, et al., (1998) J. Biol. Chem. 273,9393-9402; Xia, et al., (1998) Proc. Natl. Acad. Sci. USA 95,14196-14201; Kleuser, et al., (1998) Cancer Res. 58, 1817-1824 and Meyerzu Heringdorf, et al., (1998) EMBO J. 17, 2830-2837). Intracellular SPPin turn mobilizes calcium from internal stores independently of InsP₃(Meyer zu Heringdorf, et al., (1998) EMBO J. 17, 2830-2837 and Mattie,et al., (1994) J. Biol. Chem. 269, 3181-3188), as well as elicitingdiverse signaling pathways leading to proliferation (Rani, et al.,(1997) J. Biol. Chem. 272, 10777-10783 and Van Brocklyn, et al., (1998)J. Cell Biol. 142, 229-240.) and suppression of apoptosis (Cuvillier, etal., (1996) Nature 381, 800-803; Perez, et al., (1997) Nature Med. 3,1228-1232; Edsall, et al., (1997) J. Neurosci. 17, 6952-6960; Cuvillier,et al., (1998) J. Biol. Chem. 273, 2910-2916).

Because of its dual function as a ligand and second messenger and itspivotal role in cell growth and survival, the synthesis and degradationof SPP is expected to be tightly regulated in a spatial-temporal manner.Until recently, however, little was known of the enzymes involved in SPPmetabolism. A previous report described the purification of sphingosinekinase to apparent homogeneity from rat kidney (Olivera, et al., (1998)J. Biol. Chem. 273, 12576-12583). Subsequently the first mammaliansphingosine kinase was cloned from rat and characterized (Kohama, etal., (1998) J. Biol. Chem. 273, 23722-23728). The kinase is described asbelonging to a novel, highly conserved gene family (Kohama, et al.,(1998) J. Biol. Chem. 273, 23722-23728 and Nagiec, et al., (1998) J.Biol. Chem. 273, 19437-19442). Enforced expression of the sphingosinekinase markedly enhanced the proliferation and survival of cells,substantiating the importance of intracellularly generated SPP in cellfate decisions (Olivera, et al., (1999) J. Cell Biol. 147, 545-548).

SPP can be metabolized by two distinct pathways. In one pathway, SPP iscatabolized via a microsomal pyridoxal phosphate-dependent lyase topalmitaldehyde and phosphoethanolamine, which can then be utilized forthe biosynthesis of glycerolipids. In a second pathway, SPP isdephosphorylated by specific phosphatases to sphingosine (Spiegel, etal., (1996) FASEB J. 10, 1388-1397).

Genetic manipulation studies in yeast have demonstrated an importantrole for long-chain phosphorylated sphingoid bases in growth andsurvival of yeast after nutrient deprivation and heat stress (Mandala,et al., (1998) Proc. Nat. Acad. Sci. USA 95, 150-155; Gottlieb, et al.,(1999) Mol. Cell Biol. Res. Commun. 1, 66-71; Mao, et al., (1999)Biochem. J. 342, 667-675 and Skrzypek, et al., (1999) J. Bacteriol. 181,1134-1140) in a manner which is reminiscent of their effects onmammalian cells. Recently, the yeast genes encoding the lyase andphosphatase enzymes of these two catabolic pathways were identified inS. cerevisiae (Saba, et al. (1997) J. Biol. Chem. 272, 26087-26090;Mandala, et al., (1998) Proc. Nat. Acad. Sci. USA 95, 150-155 and Mao,et al., (1997) J. Biol. Chem. 272, 28690-28694).

The yeast SPP phosphatases encoded by LBP1 and LBP2 are members of Type2 lipid phosphate phosphohydrolases, a family of magnesium independent,membrane-bound enzymes that share sequence conservation within threedomains that are predicted to be involved in the coordination andhydrolysis of the phosphate moiety (Stukey, et al., (1997) Protein Sci.6, 469-472). A search of the yeast genome for enzymes containing thethree conserved domains revealed the presence of 4 genes encodingputative Type 2 lipid phosphatases. Two of these, DPP1 and LPP1, wereshown to encode phosphatases with activity against phosphatidic acid(PA), lysophosphatidic acid (LPA), and diacylglycerol pyrophosphate(DGPP) (Toke, et al., (1998) J. Biol. Chem. 273, 14331-14338 and Toke,et al., (1998) J. Biol. Chem. 273, 3278-3284). In contrast, LBP1 (alsoknown as YSR2 or LCB3) and LBP2 (YSR3), encode phosphatases withremarkable specificity for phosphorylated sphingoid bases and withoutactivity towards glycerolipid substrates (Mandala, et al., (1998) Proc.Nat. Acad. Sci. USA 95, 150-155; Mao, et al., (1997) J. Biol. Chem. 272,28690-28694 and Skrzypek, et al., (1999) J. Bacteriol. 181, 1134-1140).

The presence of a high affinity SPP phosphatase activity with enzymaticproperties similar to yeast SPP phosphatases has been described in cruderat liver and cerebellum extracts (De Ceuster, et al., (1995) Biochem.J. 311, 139-146). Although three isoforms of Type 2 lipid phosphatephosphohydrolases, known as LPP1/PAP2a, LPP3/PAP2b, and LPP2/PAP2c, havebeen cloned from mammalian cells (reviewed in (Brindley, et al., (1998)J. Biol. Chem. 273, 24281-24284)), these gene products appear to havebroad substrate specificity with similar efficiencies against PA, LPA,SPP, ceramide-1-P, and DGPP, when assayed in vitro in lipid/detergentmicelles.

SUMMARY OF THE INVENTION

The present invention provides polynucleotides encoding a humansphingosine-1-phosphate phosphatase(hSPP1), recombinant host cellscontaining hSPP1 polynucleotides, hSPP1 polypeptides, and methods ofusing the polynucleotides, polypeptides and host cells to conduct assaysof sphingosine-1-phosphate phosphatase activity.

The polynucleotide and polypeptide of human SPP1, an enzyme involved inthe catoblism of sphingosine-1-phosphate (SPP) are provided. Therecombinant hSPP1 enzyme is catalytically active in thedephosphorylation of SPP. The enzyme is used in in vitro and whole cellassays to screen for compounds that alter the activity of the protein orinteract with hSPP1 and, potentially, alter the expression of hSPP1. Theinvention includes the polynucleotide, protein encoded by thepolynucleotide, host cells expressing the recombinant enzyme andextracts prepared from host cells expressing the recombinant enzyme,probes and primers, and the use of these molecules in assays.

An aspect of this invention is a polynucleotide having a sequenceencoding a hSPP1 protein. In a particular embodiment the encoded proteinhas a sequence corresponding to SEQ ID NO:2. In other embodiments, theencoded protein can be a naturally occurring mutant or polymorphic formof the protein. In preferred embodiments the polynucleotide can be DNA,RNA or a mixture of both, and can be single or double stranded. Inparticular embodiments, the polynucleotide is comprised of natural,non-natural or modified nucleotides. In some embodiments, theintemucleotide linkages are linkages that occur in nature. In otherembodiments, the internucleotide linkages can be non-natural linkages ora mixture of natural and non-natural linkages. In a most preferredembodiment, the polynucleotide has the sequence contained in sequenceSEQ ID NO:1.

An aspect of this invention is a polynucleotide having a sequence of atleast about 25 contiguous nucleotides that is specific for a naturallyoccurring polynucleotide encoding a hSPP1 protein. In particularpreferred embodiments, the polynucleotides of this aspect are useful asprobes for the specific detection of the presence of a polynucleotideencoding a hSPP1 protein. In other particular embodiments, thepolynucleotides of this aspect are useful as primers for use in nucleicacid amplification based assays for the specific detection of thepresence of a polynucleotide encoding a hSPP1 protein. In preferredembodiments, the polynucleotides of this aspect can have additionalcomponents including, but not limited to, compounds, isotopes, proteinsor sequences for the detection of the probe or primer.

An aspect of this invention is an expression vector including apolynucleotide encoding a hSPP1 protein, or a complementary sequence,and regulatory regions. In a particular embodiment the encoded proteinhas a sequence corresponding to SEQ ID NO:2. In particular embodiments,the vector can have any of a variety of regulatory regions known andused in the art as appropriate for the types of host cells the vectorcan be used in. In a most preferred embodiment, the vector hasregulatory regions appropriate for the expression of the encoded proteinin mammalian host cells. In other embodiments, the vector has regulatoryregions appropriate for expression of the encoded protein in othereukaryotes, bacteria, yeasts, insect cells, cyanobacteria oractinomycetes. In some preferred embodiments the regulatory regionsprovide for inducible expression while in other preferred embodimentsthe regulatory regions provide for constitutive expression. Finally,according to this aspect, the expression vector can be derived from aplasmid, phage, virus or a combination thereof.

An aspect of this invention is host cell comprising an expression vectorincluding a polynucleotide encoding a hSPP1 protein, or a complementarysequence, and regulatory regions. In a particular embodiment the encodedprotein has a sequence corresponding to SEQ ID NO:2. In preferredembodiments, the host cell is a eukaryote, yeast, insect cell,gram-positive bacterium, cyanobacterium or actinomycete. In a mostpreferred embodiment, the host cell is a mammalian cell.

An aspect of this invention is a process for expressing a hSPP1 proteinin a host cell. In this aspect a host cell is transformed or transfectedwith an expression vector including a polynucleotide encoding a hSPP1protein, or a complementary sequence. According to this aspect, the hostcell is cultured under conditions conducive to the expression of theencoded hSPP1 protein. In particular embodiments the expression isinducible or constitutive. In a particular embodiment the encodedprotein has a sequence corresponding to SEQ ID NO:2.

An aspect of this invention is a purified hSPP1 polypeptide having anamino acid sequence of SEQ ID NO:2 or the sequence of a naturallyoccurring mutant or polymorphic form of the protein.

An aspect of this invention is a method of determining whether acandidate compound can alter the activity of a hSPP1 polypeptide.According to this aspect a polynucleotide encoding the polypeptide isused to construct an expression vector appropriate for a particular hostcell. The host cell is transformed or transfected with the expressionvector and cultured under conditions conducive to the expression of thehSPP1 polypeptide. The cell is contacted with the candidate. Finally,one measures the activity of the hSPP1 polypeptide in the presence ofthe candidate. If the activity is lower relative to the activity of theprotein in the absence of the candidate, then the candidate is ainhibitor of the hSPP1 polypeptide. In preferred embodiments, thepolynucleotide encodes a protein having an amino acid sequence of SEQ IDNO:2 or a naturally occurring mutant of polymorphic form thereof. Inother preferred embodiments, the polynucleotide has the sequence of SEQID NO: 1. In particular embodiments, the relative activity of hSPP1 isdetermined by comparing the activity of the hSPP1 in a host cell. Insome embodiments, the host cell is disrupted and the candidate iscontacted to the released membrane extract. In other embodiments, thecells can be disrupted contacting with the candidate and beforedetermining the activity of the hSPP1 protein. Finally, according tothis aspect the relative activity can determined by comparison to apreviously measured or expected activity value for the hSPP1 activity inthe host under the conditions. However, in preferred embodiments, therelative activity is determined by measuring the activity of the hSPP1in a control cell that was not contacted with a candidate compound. Inparticular embodiments, the host cell is a mammalian cell and theprotein inhibited is the hSPP1 produced by the mammalian cell.

By “about” it is meant within 10% to 20% greater or lesser thanparticularly stated.

As used herein an “agonist” is a compound or molecule that interactswith and stimulates an activity of hSPP1.

As used herein an “antagonist” is a compound that interacts with hSPP1and interferes with the interaction of hSPP1 and SPP.

As used herein an “inhibitor” is a compound that interacts with andinhibits or prevents hSPP1 from catalyzing the dephosphorylation of SPP.

As used herein a “modulator” is a compound that interacts with an aspectof cellular biochemistry to effect an increase or decrease in the amountof a polypeptide of hSPP1 present in, at the surface or in the periplasmof a cell, or in the surrounding serum or media. The change in amount ofthe hSPP1 polypeptide can be mediated by the effect of a modulator onthe expression of the protein, e.g., the transcription, translation,post-translational processing, translocation or folding of the protein,or by affecting a component(s) of cellular biochemistry that directly orindirectly participates in the expression of the protein. Alternatively,a modulator can act by accelerating or decelerating the turnover of theprotein either by direct interaction with the protein or by interactingwith another component(s) of cellular biochemistry which directly orindirectly effects the change.

An aspect of this invention is a transgenic animal useful for the studyof the tissue and temporal specific expression or activity of the hSPP1gene in a non-human animal. The animal is also useful for studying theability of a variety of compounds to act as agonists, antagonists orinhibitors of hSPP1 activity or expression in vivo or, by providingcells for culture or assays, in vitro. In an embodiment of this aspectof the invention, the animal is used in a method for the preparation ofa further animal which lacks a functional endogenous hSPP1 gene. Inanother embodiment, the animal of this aspect is used in a method toprepare an animal which expresses a hSPP1 gene in the absence of theexpression of a endogenous gene. In particular embodiments the non-humananimal is a mouse. In further embodiments the hSPP1 gene is a wild-typehSPP1 gene or a mutant hSPP1 gene.

All of the references cited herein are incorporated by reference intheir entirety as background material.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the polynucleotide sequence of SEQ ID NO:1.

FIG. 2 is the polypeptide sequence of SEQ ID NO:2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a polynucleotide and polypeptide of ahuman sphingosine-1-phosphate phosphatase, referred to herein as hSPP1.The polynucleotide and polypeptide are used to further provideexpression vectors, host cells comprising the vectors, probes andprimers, antibodies against the hSPP1 protein and polypeptides thereof,assays for the presence or expression of hSPP1 and assays for theidentification of compounds that interact with hSPP1.

Polynucleotides

Polynucleotides useful in the present invention include those describedherein and those that one of skill in the art will be able to derivetherefrom following the teachings of this specification. A preferredaspect of the present invention is a recombinant polynucleotide encodinga human SPP1 protein. One preferred embodiment is a nucleic acid havingthe sequence disclosed in FIG. 1, SEQ ID NO:1 and disclosed as follows:

cggcccgcct tcccgggggt tccgttatca tgtcgctgag gcagcgcctg (SEQ ID NO: 1)gcccagctgg ttggccgtct gcaggacccg cagaaagtgg cccgtttcca gcggctgtgcggggtggaag cgccgccgcg ccgctcagca gaccggaggg aggatgagaa agcggaggcgcctctcgccg gagaccctcg actgcgaggg cggcagccag gggcgcctgg aggcccccagcctcccggga gcgaccgcaa tcagtgcccg gccaagccgg acggcggcgg cgcccccaacggcgtgcgga acgggctggc ggccgagctg ggcccggcct cgccgcggcg cgcgggcgctctgcgccgca actcgctgac gggcgaggag ggccagctgg cccgcgtgag caactggccgctctactgcc tgttctgctt cggcacggag ctgggcaacg aactcttcta catcctgttcttccccttct ggatctggaa cctggaccct ctggtgggcc ggaggctcgt ggtcatctgggtgctggtca tgtacctggg ccagtgcacc aaggacatca tccgctggcc gaggcccgcctcgccgcccg tggtcaagtt ggaggtcttc tacaactctg agtacagcat gccctccacccatgccatgt ccggcaccgc catccccatt tctatggtcc tcctcaccta tggccgctggcagtaccctc ttatatatgg actgattctt attccctgct ggtgttctct agtttgcctaagtagaattt acatgggaat gcactctatt ctggatatta ttgctggatt cctatataccattttaatct tagctgtctt ctatccattt gtggacctga ttgacaactt caaccaaactcacaaatatg ctccattcat catcatcggg cttcatttag ctttggggat cttttctttcactcttgaca cctggagcac atcccgagga gacacagccg agatactagg aagtggtgctggaattgcat gtggatctca tgttacttat aacatgggtc tagtattaga tccttctctagatacattac ctttagctgg gccccccatt actgtgactc tgtttggaaa agccatattgcggatcctca tagggatggt atttgtacta ataatcagag atgtaatgaa aaagatcaccattcctttag cctgcaaaat cttcaatata ccgtgtgatg atattcgaaa agcaagacagcacatggaag ttgaacttcc ttatcggtat attacctatg gaatggttgg tttctccatcacattttttg ttccttacat atttttcttt attggtatct cttgatggag aagtattgtttatgataaga aaggagggta tcagttactg atacccaaaa atatattcca

The translation initiation and termination codons are underlined. Aparticularly preferred embodiment is a polynucleotide comprising thecoding sequence of hSPP1 of SEQ ID NO:1.

The isolated nucleic acid molecules of the present invention can includea ribonucleic or deoxyribonucleic acid molecule, which can be single(coding or noncoding strand) or double stranded, as well as syntheticnucleic acid, such as a synthesized, single stranded polynucleotide.

The present invention also relates to recombinant vectors andrecombinant hosts, both prokaryotic and eukaryotic, which contain thesubstantially purified nucleic acid molecules disclosed throughout thisspecification.

As used herein a “polynucleotide” is a nucleic acid of more than onenucleotide. A polynucleotide can be made up of multiple polynucleotideunits that are referred to by description of the unit. For example, apolynucleotide can comprise within its bounds a polynucleotide(s) havinga coding sequence(s), a polynucleotide(s) that is a regulatory region(s)and/or other polynucleotide units commonly used in the art.

An “expression vector” is a polynucleotide having regulatory regionsoperably linked to a coding region such that, when in a host cell, theregulatory regions can direct the expression of the coding sequence. Theuse of expression vectors is well known in the art. Expression vectorscan be used in a variety of host cells and, therefore, the regulatoryregions are preferably chosen as appropriate for the particular hostcell.

A “regulatory region” is a polynucleotide that can promote or enhancethe initiation or termination of transcription or translation of acoding sequence. A regulatory region includes a sequence that isrecognized by the RNA polymerase, ribosome, or associated transcriptionor translation initiation or termination factors of a host cell.Regulatory regions that direct the initiation of transcription ortranslation can direct constitutive or inducible expression of a codingsequence.

Polynucleotides of this invention contain full length or partial lengthsequences of the hSPP1 gene sequences disclosed herein. Polynucleotidesof this invention can be single or double stranded. If single stranded,the polynucleotides can be a coding, “sense,” strand or a complementary,“antisense,” strand. Antisense strands can be useful as modulators ofthe gene by interacting with RNA encoding the hSPP1 protein. Antisensestrands are preferably less than full length strands having sequencesunique or specific for RNA encoding the hSPP1 protein.

The polynucleotides can include deoxyribonucleotides, ribonucleotides ormixtures of both. The polynucleotides can be produced by cells, incell-free biochemical reactions or through chemical synthesis.Non-natural or modified nucleotides, including inosine, methyl-cytosine,deaza-guanosine, etc., can be present. Natural phosphodiesterinternucleotide linkages can be appropriate. However, polynucleotidescan have non-natural linkages between the nucleotides. Non-naturallinkages are well known in the art and include, without limitation,methylphosphonates, phosphorothioates, phosphorodithionates,phosphoroamidites and phosphate ester linkages. Dephospho-linkages arealso known, as bridges between nucleotides. Examples of these includesiloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, andthioether bridges. “Plastic DNA,” having, for example, N-vinyl,methacryloxyethyl, methacrylamide or ethyleneimine internucleotidelinkages, can be used. “Peptide Nucleic Acid” (PNA) is also useful andresists degradation by nucleases. These linkages can be mixed in apolynucleotide.

As used herein, “purified” and “isolated” are utilized interchangeablyto stand for the proposition that the polynucleotide, protein andpolypeptide, or respective fragments thereof in question have beenremoved from the in vivo environment so that they exist in a form orpurity not found in nature. Purified or isolated nucleic acid moleculescan be manipulated by the skilled artisan, such as but not limited tosequencing, restriction digestion, site-directed mutagenesis, andsubcloning into expression vectors for a nucleic acid fragment as wellas obtaining the wholly or partially purified protein or proteinfragment so as to afford the opportunity to generate polyclonalantibodies, monoclonal antibodies, or perform amino acid sequencing orpeptide digestion. Therefore, the nucleic acids claimed herein can bepresent in whole cells or in cell lysates or in a partially orsubstantially purified form. It is preferred that the molecule bepresent at a concentration at least about five-fold to ten-fold higherthan that found in nature. A polynucleotide is considered substantiallypure if it is obtained purified from cellular components by standardmethods at a concentration of at least about 100-fold higher than thatfound in nature. A polynucleotide is considered essentially pure if itis obtained at a concentration of at least about 1000-fold higher thanthat found in nature. We most prefer polynucleotides that have beenpurified to homogeneity, that is, at least 10,000-100,000 fold. Achemically synthesized nucleic acid sequence is considered to besubstantially purified when purified from its chemical precursors by thestandards stated above.

The term “recombinant” is used to denote those polynucleotidepreparations, constructs, expressions systems and cell lines containingthe same which are made by the hand of man.

Included in the present invention are assays that employ further novelpolynucleotides that hybridize to hSPP1 sequences under stringentconditions. By way of example, and not limitation, a procedure usingconditions of high stringency is as follows: Prehybridization of filterscontaining DNA is carried out for 2 hr. to overnight at 65° C. in buffercomposed of 6× SSC, 5× Denhardt's solution, and 100 μg/ml denaturedsalmon sperm DNA. Filters are hybridized for 12 to 48 hrs at 65° C. inprehybridization mixture containing 100 μg/ml denatured salmon sperm DNAand 5-20×10⁶ cpm of ³²P-labeled probe. Washing of filters is done at 37°C. for 1 hr in a solution containing 2× SSC, 0.1% SDS. This is followedby a wash in 0.1× SSC, 0.1% SDS at 50° C. for 45 min. beforeautoradiography.

Other procedures using conditions of high stringency would includeeither a hybridization step carried out in 5× SSC, 5× Denhardt'ssolution, 50% formamide at 42° C. for 12 to 48 hours or a washing stepcarried out in 0.2× SSPE, 0.2% SDS at 65° C. for 30 to 60 minutes.

Reagents mentioned in the foregoing procedures for carrying out highstringency hybridization are well known in the art. Details of thecomposition of these reagents can be found in, e.g., Sambrook, et al.,1989, Molecular Cloning: A Laboratory Manual, second edition, ColdSpring Harbor Laboratory Press. In addition to the foregoing, otherconditions of high stringency which may be used are well known in theart.

“Identity” is a measure of the identity of nucleotide sequences or aminoacid sequences. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. See, e.g.,:(COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed. Oxford UniversityPress, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS,Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OFSEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds. HumanaPress, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, vonHeinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).While there exist a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H., and Lipton, D., SIAM J AppliedMath (1988)48:1073). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCG program package (Devereux, J. et a., Nucleic AcidsResearch (1984)12(1):387), BLAST?, BLASTN, FASTA (Atschul, S. F. et al.,J Molec Biol (1990)215:403).

As an illustration, by a polynucleotide having a nucleotide sequencehaving at least, for example, 95% “identity” to a reference nucleotidesequence of SEQ ID NO:1, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five differences per each100 nucleotides of the reference nucleotide sequence of SEQ ID NO:1. Inother words, to obtain a polynucleotide having a nucleotide sequence atleast 95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These mutations of the reference sequence may occur at the 5′or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among nucleotides in the reference sequence or in one ormore contiguous groups within the reference sequence.

Similarly, by a polypeptide having an amino acid sequence having atleast, for example, 95% identity to a reference amino acid sequence ofSEQ ID NO:2 is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of SEQ ID NO:2. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a reference amino acid sequence, up to 5% of the amino acidresidues in the reference sequence may be deleted or substituted withanother amino acid, or a number of amino acids up to 5% of the totalamino acid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence of anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

Polypeptides

A preferred aspect of the present invention is a substantially purifiedform of the human SPP1 protein. A preferred embodiment is a protein thathas the amino acid sequence which is shown in FIG. 2, in SEQ ID NO:2 anddisclosed in single letter code as follows:

MSLRQRLAQL VGRLQDPQKV ARFQRLCGVE APPRRSADRR EDEKAEAPLA GDPRLRGRQP (SEQID NO: 2) GAPGGPQPPG SDRNQCPAKP DGGGAPNGVR NGLAAELGPA SPRRAGALRRNSLTGEEGQL ARVSNWPLYC LFCFGTELGN ELFYILFFPF WIWNLDPLVG RRLVVIWVLVMYLGQCTKDI IRWPRPASPP VVKLEVFYNS EYSMPSTHAM SGTAIPISMV LLTYGRWQYPLIYGLILIPC WCSLVCLSRI YMGMHSILDI IAGFLYTILI LAVFYPFVDL IDNFNQTHKYAPFIIIGLHL ALGIFSFTLD TWSTSRGDTA EILGSGAGIA CGSHVTYNMG LVLDPSLDTLPLAGPPITVT LFGKAILRIL IGMVFVLIIR DVMKKITIPL ACKIFNIPCD DIRKARQHMEVELPYRYITY GMVGFSITFF VPYIFFFIGI S*

The present invention also relates to biologically active fragments andmutant or polymorphic forms of the hSPP1 polypeptide sequence set forthas SEQ ID NO:2, including but not limited to amino acid substitutions,deletions, additions, amino terminal truncations and carboxy-terminaltruncations such that these mutations provide for proteins or proteinfragments of diagnostic, therapeutic or prophylactic use and would beuseful for screening for modulators, and/or inhibitors of hSPP1function.

Using the disclosure of polynucleotide and polypeptide sequencesprovided herein to isolate polynucleotides encoding naturally occurringforms of hSPP1, one of skill in the art can determine whether suchnaturally occurring forms are mutant or polymorphic forms of hSPP1 bysequence comparison. One can further determine whether the encodedprotein, or fragments of any hSPP1 protein, are biologically active byroutine testing of the protein or fragment in a in vitro or in vivoassay for the biological activity of the hSPP1 protein. For example, onecan express N-terminal or C-terminal truncations, or internal additionsor deletions, in host cells and test for their ability to catalyze thedephosphorylation of sphingosine-1-phosphate.

It is known that there is a substantial amount of redundancy in thevarious codons which code for specific amino acids. Therefore, thisinvention is also directed to those DNA sequences that encode RNAcomprising alternative codons which code for the eventual translation ofthe identical amino acid.

Therefore, the present invention discloses codon redundancy which canresult in different DNA molecules encoding an identical protein. Forpurposes of this specification, a sequence bearing one or more replacedcodons will be defined as a degenerate variation. Also included withinthe scope of this invention are mutations either in the DNA sequence orthe translated protein which do not substantially alter the ultimatephysical properties of the expressed protein. For example, substitutionof valine for leucine, arginine for lysine, or asparagine for glutaminemay not cause a change in functionality of the polypeptide. However, anygiven change can be examined for any effect on biological function bysimply assaying for the ability to catalyze the catabolism ofsphingosine-1-phosphate as compared to an unaltered hSPP1 protein.

It is known that DNA sequences coding for a peptide can be altered so asto code for a peptide having properties that are different than those ofthe naturally occurring peptide. Methods of altering the DNA sequencesinclude but are not limited to site directed mutagenesis. Examples ofaltered properties include but are not limited to changes in theaffinity of an enzyme for a substrate.

As used herein, a “biologically active equivalent” or “functionalderivative” of a wild-type mSPP1 possesses a biological activity that issubstantially similar to the biological activity of a wild type mSPP1.The term “functional derivative” is intended to include the “fragments,”“mutants,” “variants,” “degenerate variants,” “analogs,” “orthologues,”and “homologues” and “chemical derivatives” of a wild type mSPP1 proteinthat can catalyze the catabolism of sphingosine-1-phosphate.

The term “fragment” refers to any polypeptide subset of wild-type hSPP1.The term “mutant” is meant to refer to a molecule that may besubstantially similar to the wild-type form but possesses distinguishingbiological characteristics. Such altered characteristics include but arein no way limited to altered substrate binding, altered substrateaffinity and altered sensitivity to chemical compounds affectingbiological activity of the hSPP1. The term “variant” refers to amolecule substantially similar in structure and function to either theentire wild-type protein or to a fragment thereof. A molecule is“substantially similar” to a wild-type hSPP1-like protein if bothmolecules have substantially similar structures or if both moleculespossess similar biological activity. Therefore, if the two moleculespossess substantially similar activity, they are considered to bevariants even if the exact structure of one of the molecules is notfound in the other or even if the two amino acid sequences are notidentical. The term “analog” refers to a molecule substantially similarin function to either the full-length hSPP1 protein or to a biologicallyactive fragment thereof.

As used herein in reference to a hSPP1 gene or encoded protein, a“polymorphic” hSPP1 is a hSPP1 that is naturally found in the populationof animals at large. Typically, the genes for polymorphs of hSPP1 can bedetected by high stringency hybridization using the hSPP1 gene as aprobe. A polymorphic form of hSPP1 can be encoded by a nucleotidesequence different from the particular hSPP1 gene disclosed herein asSEQ ID NO:1. However, because of silent mutations, a polymorphic hSPP1gene can encode the same or different amino acid sequence as thatdisclosed herein. Further, some polymorphic forms hSPP1 will exhibitbiological characteristics that distinguish the form from wild-typehSPP1 activity, in which case the polymorphic form is also a mutant.

A protein or fragment thereof is considered purified or isolated when itis obtained at least partially free from it's natural environment in acomposition or purity not found in nature. It is preferred that themolecule be present at a concentration at least about five-fold toten-fold higher than that found in nature. A protein or fragment thereofis considered substantially pure if it is obtained at a concentration ofat least about 100-fold higher than that found in nature. A protein orfragment thereof is considered essentially pure if it is obtained at aconcentration of at least about 1000-fold higher than that found innature. It is most prefer proteins that have been purified tohomogeneity, that is, at least 10,000-100,000 fold.

The term “recombinant” with respect to a polypeptide of the presentinvention refers only to polypeptides that are made by recombinantprocesses, expressed by recombinant cells or purified from natural cellsas described above. Preparations having partially purified hSPP1polypeptide are meant to be within the scope of the term “recombinant.”

Expression of hSPP1

A variety of expression vectors can be used to express recombinant hSPP1polypeptide in host cells. Expression vectors are defined herein asnucleic acid sequences that include regulatory sequences for thetranscription of cloned DNA and the translation of their mRNAs in anappropriate host. Such vectors can be used to express genes in a varietyof hosts such as yeast, bacteria, bluegreen algae, plant cells, insectcells and animal cells. Specifically designed vectors allow theshuttling of genes between hosts such as bacteria-yeast orbacteria-animal cells. An appropriately constructed expression vectorshould contain: an origin of replication for autonomous replication inhost cells, selectable markers, a limited number of useful restrictionenzyme sites, a potential for high copy number, and regulatorysequences. A promoter is defined as a regulatory sequence that isinvolved in the binding of RNA polymerase to DNA and the initiation ofRNA synthesis. A strong promoter is one which causes mRNAs to beinitiated at high frequency. Expression vectors can include, but are notlimited to, cloning vectors, modified cloning vectors, specificallydesigned plasmids or viruses.

In particular, a variety of bacterial expression vectors can be used toexpress recombinant hSPP1 in bacterial cells. Commercially availablebacterial expression vectors which are suitable for recombinant hSPP1expression include, but are not limited to pQE (QIAGEN), pET11a orpET15b (NOVAGEN), lambda gt11 (INVITROGEN), and pKK223-3 (PHARMACIA).

Alternatively, one can express hSPP1 DNA in cell-freetranscription-translation systems, or hSPP1 RNA in cell-free translationsystems. Cell-free synthesis of hSPP1 polypeptide can be in batch orcontinuous formats known in the art.

One can also synthesize hSPP1 chemically, although this method is notpreferred.

A variety of host cells can be employed with expression vectors tosynthesize hSPP1 protein. These can include E. coli, Bacillus, andSalmonella. Insect and yeast cells can also be appropriate. However, themost preferred host cell is a mammalian host cell.

Following expression of hSPP1 in a host cell, hSPP1 polypeptides can berecovered. Several protein purification procedures are available andsuitable for use. hSPP1 protein and polypeptides can be purified fromcell lysates and extracts, or from culture medium, by variouscombinations of, or individual application of methods includingdetergent solubilization, ultrafiltration, acid extraction, alcoholprecipitation, salt fractionation, ionic exchange chromatography,phosphocellulose chromatography, lecithin chromatography, affinity(e.g., antibody or His-Ni) chromatography, size exclusionchromatography, hydroxylapatite adsorption chromatography andchromatography based on hydrophobic or hydrophillic interactions. Insome instances, protein denaturation and refolding steps can beemployed. High performance liquid chromatography (HPLC) and reversedphase HPLC can also be useful. Dialysis can be used to adjust the finalbuffer composition.

The hSPP1 protein itself is useful in assays to identify compounds thatalter the activity of the protein—including compounds that inhibit orstimulate the activity of the protein. The hSPP1 protein is also usefulfor the generation of antibodies against the protein, structural studiesof the protein, and structure/function relationships of the protein.

Modulators, Agonist, Antagonists and Inhibitors of hSPP1

The present invention is also directed to methods for screening forcompounds which modulate the expression of, stimulate or inhibit theactivity of a hSPP1 protein. Compounds which modulate or inhibit hSPP1can be DNA, RNA, peptides, proteins, or non-proteinaceous organic orinorganic compounds or other types of molecules. Compounds that modulatethe expression of DNA or RNA encoding hSPP1 or are agonists, antagonistsor inhibitors of the biological function of hSPP1 can be detected by avariety of assays. The assay can be a simple “yes/no” assay to determinewhether there is a change in expression or activity. The assay can bemade quantitative by comparing the expression or activity of a testsample with the level or degree of expression or activity in a standardsample, that is, a control. A compound that is a modulator can bedetected by measuring the amount of the mRNA and/or hSPP1 produced inthe presence of the compound. A compound that is an agonist, antagonistor inhibitor can be detected by measuring the specific activity of thehSPP1 protein in the presence and absence of the compound.

The proteins, DNA molecules, RNA molecules and antibodies lendthemselves to the formulation of kits suitable for the detection andanalysis of hSPP1. Such a kit would comprise a compartmentalized carriersuitable to hold in close confinement at least one container. Thecarrier would further comprise reagents such as recombinant hSPP1 oranti-hSPP1 antibodies suitable for detecting hSPP1. The carrier can alsocontain a means for detection such as labeled antigen or enzymesubstrates or the like.

Assays

Assays of the present invention can be designed in many formatsgenerally known in the art of screening compounds for biologicalactivity or for binding to enzymes. Assays of the present invention canadvantageously exploit the activity of hSPP1 in converting SPP tosphingosine or dihydrosphingosine-1-phosphate (DHSP) todihydrosphingosine (DHS). For convenience, the description that followswill refer mostly to the conversion of SPP to sphingosine, however,either conversion can be followed in an assay.

The present invention includes methods of identifying compounds thatspecifically interact with hSPP1 polypeptides. Compounds that interactwith the enzyme can stimulate or inhibit the activity of hSPP1. Thespecificity of binding of compounds having affinity for hSPP1 can beshown by measuring the affinity of the compounds to membranes fromrecombinant cells expressing a hSPP1 polypeptide. Expression of hSPP1polypeptides and screening for compounds that bind to hSPP1 or thatinhibit the conversion of SPP to sphingosine, provides an effectivemethod for the rapid selection of compounds with affinity for hSPP1. TheSPP can be radiolabeled but can also be labeled by other means known inthe art and thereafter can be used to follow the conversion of thelabeled SPP to sphingosine in assays of hSPP1 activity.

If one desires to produce a fragment of the hSPP1 or mutant, polymorphicor allelic variants of the hSPP1, one can test those products in theassays described below and compare the results to those obtained usingan active hSPP1 polypeptide of SEQ ID NO:2. In this manner one caneasily assess the ability of the fragment, mutant, polymorph or allelicvariant to bind compounds, be activated by agonists or be inactivated orinhibited by antagonists of hSPP1.

Therefore, the present invention includes assays by which compounds thatare hSPP1 agonists, antagonists, and inhibitors may be identified. Theassay methods of the present invention differ from those described inthe art because the present assays incorporate at least one step whereinthe interaction of SPP and an hSPP1 polypeptide, preferably arecombinant polypeptide, is incorporated into the assay.

General methods for identifying ligands, agonists and antagonists arewell known in the art and can be adapted to identify agonists andantagonists of hSPP1. The order of steps in any given method can bevaried or performed concurrently as will be recognized by those of skillin the art of assays. The following is a sampling of the variety offormats that can be used to conduct an assay of the present invention.

Accordingly, the present invention includes a method for determiningwhether a candidate compound is an agonist or an inhibitor of hSPP1, themethod of which comprises:

(a) transfecting cells with an expression vector encoding a hSPP1polypeptide;

(b) allowing the transfected cells to grow for a time sufficient toallow hSPP1 to be expressed in the cells;

(c) exposing portions of the cells to labeled SPP in the presence and inthe absence of the compound;

(d) measuring the conversion of the labeled SPP to sphingosine in theportions of cells; and

(e) comparing the amount of conversion of SPP to sphingosine in thepresence and the absence of the compound where a decrease in the amountof conversion of SPP to sphingosine in the presence of the compoundindicates that the compound is an inhibitor of hSPP1 whereas an increasein the conversion of SPP to sphingosine indicates that the compound isan agonist of hSPP1.

The conditions under which step (c) of the method is practiced areconditions that are typically used in the art for the study ofprotein-ligand interactions: e.g., physiological pH; salt conditionssuch as those represented by such commonly used buffers as PBS or intissue culture media; a temperature of about 4° C. to about 55° C. Inthis step the SPP and candidate compound can be applied to the cellsequentially or concurrently. It is preferred that the compound isapplied first or that the compound and SPP are applied concurrently.

The above whole cell methods can be used in assays where one desires toassess whether a compound can traverse a cell membrane to interact withhSPP1. However, the above methods can be modified in that, rather thanexposing the test cells to the candidate compound, membranes can beprepared from the cells and those membranes can be exposed to thecompound. Such a modification utilizing membranes rather than cells iswell known in the art and is described in, e.g., Hess et al., 1992.Particular methods of assaying membranes are described in the Examplesbelow.

Accordingly, the present invention provides a method of using theinteraction of SPP and hSPP1 for determining whether a candidatecompound is an agonist or inhibitor of a hSPP1 polypeptide in membranescomprising:

(a) providing test cells by transfecting cells with an expression vectorthat directs the expression of hSPP1 in the cells;

(b) preparing membranes containing hSPP1 from the test cells;

(c) exposing the membranes to SPP under conditions such that the ligandbinds to the polypeptide in the membranes;

(d) further exposing the membranes to a candidate compound under similarconditions;

(e) measuring the amount of conversion of SPP to sphingosine in themembranes in the presence and the absence of the compound;

(f) comparing the amount of conversion of SPP to sphingosine in thepresence and the absence of the compound where a decrease in the amountof conversion of SPP to sphingosine in the presence of the compoundindicates that the compound is an inhibitor of hSPP1; whereas anincrease in the conversion of SPP to sphingosine indicates that thecompound is an agonist of hSPP1.

As a further modification of the above-described methods, RNA encodinghSPP1 can be prepared as, e.g., by in vitro transcription using aplasmid containing hSPP1 under the control of a bacteriophage 17promoter, and the RNA can be microinjected into Xenopus oocytes in orderto cause the expression of hSPP1 in the oocytes. Compounds are thentested for binding to the hSPP1 or inhibition of activity of hSPP1expressed in the oocytes. As in all assays of this invention, a stepusing the interaction of SPP and hSPP1 is incorporated into the assay.

Transgenic Animals

In reference to the transgenic animals of this invention, we refer totransgenes and genes. As used herein, a “transgene” is a geneticconstruct including a gene. The transgene is typically integrated intoone or more chromosomes in the cells in an animal or its ancestor bymethods known in the art. Once integrated, the transgene is carried inat least one place in the chromosomes of a transgenic animal. A gene isa nucleotide sequence that encodes a protein. The gene and/or transgenecan also include genetic regulatory elements and/or structural elementsknown in the art.

The term “animal” is used herein to include all mammals, except humans.It also includes an individual animal in all stages of development,including embryonic and fetal stages. Preferably the animal is a rodent,and most preferably mouse or rat. A “transgenic animal” is an animalcontaining one or more cells bearing genetic information received,directly or indirectly, by deliberate genetic manipulation at asubcellular level, such as by microinjection or infection withrecombinant virus. This introduced DNA molecule can be integrated withina chromosome, or it can be extra-chromosomally replicating DNA. Unlessotherwise noted or understood from the context of the description of ananimal, the term “transgenic animal” as used herein refers to atransgenic animal in which the genetic information was introduced into agerm line cell, thereby conferring the ability to transfer theinformation to offspring. If offspring in fact possess some or all ofthe genetic information, then they, too, are transgenic animals. Thegenetic information is typically provided in the form of a transgenecarried by the transgenic animal.

The genetic information received by the non-human animal can be foreignto the species of animal to which the recipient belongs, or foreign onlyto the particular individual recipient. In the latter case, theinformation can be altered or it can be expressed differently than thenative gene of the animal. Alternatively, the altered or introduced genecan cause the native gene to become non-functional.

As used herein, a “targeted gene” or “Knockout” (KO) transgene is a DNAsequence introduced into the germline of a non-human animal by way ofhuman intervention, including but not limited to, the methods describedherein. The targeted genes of the invention include nucleic acidsequences which are designed to specifically alter cognate endogenousalleles of the non-human animal.

An altered hSPP1 gene should not fully encode the same proteinendogenous to the host animal, and its expression product can be alteredto a minor or great degree, or absent altogether. In cases where it isuseful to express a non-native hSPP1 protein in a transgenic animal inthe absence of a endogenous hSPP1 protein we prefer that the alteredhSPP1 gene induce a null, “knockout,” phenotype for the native gene ofthe animal. However a more modestly modified hSPP1 gene can also beuseful and is within the scope of the present invention.

A type of target cell for transgene introduction is the embryonic stemcell (ES). ES cells can be obtained from pre-implantation embryoscultured in vivo and fused with embryos (M. J. Evans et al., Nature292:154-156(1981); Bradley et al., Nature 309:255-258 (1984); Gossler etal. Proc. Natl. Acad. Sci. USA 83:9065-9069 (1986); and Robertson etal., Nature 322:445-448 (1986)). Transgenes can be efficientlyintroduced into the ES cells by a variety of standard techniques such asDNA transfection, microinjection, or by retrovirus-mediatedtransduction. The resultant transformed ES cells can thereafter becombined with blastocysts from a non-human animal. The introduced EScells thereafter colonize the embryo and contribute to the germ line ofthe resulting chimeric animal (R. Jaenisch, Science 240: 1468-1474(1988)). Animals are screened for those resulting in germlinetransformants. These are crossed to produce animals homozygous for thetransgene.

Methods for evaluating the targeted recombination events as well as theresulting knockout mice are readily available and known in the art. Suchmethods include, but are not limited to DNA (Southern) hybridization todetect the targeted allele, polymerase chain reaction (PCR),polyacrylamide gel electrophoresis (PAGE) and Western blots to detectDNA, RNA and protein.

This may have a therapeutic aim. The presence of a mutant, allele orvariant sequence within cells of an organism, particularly when in placeof a homologous endogenous sequence, may allow the organism to be usedas a model in testing and/or studying the role of the hSPP1 gene orsubstances which modulate activity of the encoded polypeptide and/orpromoter in vivo or are otherwise indicated to be of therapeuticpotential.

EXAMPLE 1

Identification of Human Homologs to Mouse Sphingosine-1-PhosphatePhosphatases

To identify human homologs of the mouse sphingosine-1-phosphate (SPP)phosphatase encoded by mSPP1, the EST database was searched using theTBLASTN algorithm. Several human sequences in the EST database wereidentified with high homology to mSPP1 including gb:AA376229,gb:AA331563, gb:AA371461, gb:AA375349, gb:AA133909, gb:AA805328, andgsc:00005190.

Bacterial cultures transformed with these EST clones were obtained fromATCC or Merck Genome Sequencing Center and plasmid DNAs were preparedusing WIZARD DNA Purification System (PROMEGA). DNA preparations weresubjected to automated sequence analysis using the PRISM Dye Deoxyterminator cycle sequencing kit (APPLIED BIOSYSTEMS, Foster City,Calif.) on an ABI PRISM 377 instrument. T7 sequencing primercomplementary to the vector was used in sequencing reactions. Databasesearches (GENBANK, EMBL, SWISS-PROTEIN, PIR, DEST) sequence alignments,and analysis of the nucleotide and protein sequences were carried outusing TBLAST, the GCG Sequence Analysis Software Package (Madison, Wis.)and VectorNTI Contig Express. Sequence alignments confirmed the homologyto mSPP1.

EXAMPLE 2

PCR Amplification of the hSPP1 3′ end

A RACE (Rapid Amplification of cDNA Ends) methodology was initiallyemployed to clone the human SPP phosphatase gene from cDNA libraries.Gene specific primers were designed based on the human ESTs AA376229,AA331563, AA371461, AA375349, AA133909 and AA805328. The gene specificprimers had a 50-70% GC content and Tm≦70° C. Primers were paired withadapter primers and used to amplify the 5′ and 3′ ends using adapterligated double stranded cDNAs and PCR kits purchased from CLONTECH(Marathon-Ready human placental cDNA, Marathon cDNA Amplification Kit,and Advantage PCR polymerase). An APPLIED BIOSYSTEMS GENEAMP PCR 9700instrument was used with cycling conditions of: 94° C. for 1 min; 5cycles of 94° C. for 30 sec and 72° C. for 4 min; 5 cycles of 94° C. for30 sec and 70° C. for 4 min; 30 cycles of 94° C. for 30 sec, 68° C. for4 min; 70° C. for 7 min; and then hold at 4° C.

Primers 5′-CACCGCCATCCCCATTTCTATGG (SEQ ID NO:3) and5′-GTGCTCCAGGTGTCAAGAGTGAAAG (SEQ ID NO:4)were used to amplify a 443 bp fragment from human fetal brain cDNA. ThePCR product was isolated by GENECLEAN (BIO101) and ligated into pCR2.1using a TA cloning kit (INVITROGEN) to make plasmid SPP(A)-pCR2.1.

Primers 5′-GGAAGTGGTGCTGGAATTGCATG (SEQ ID NO:5) and5′-GCCTCCCATGTTCAACATCATGG (SEQ ID NO:6)were used to amplify a 930 base pair fragment from human placental cDNA.The PCR product was isolated by GENECLEAN (BIO101) and ligated intopCR2.1 to make plasmid SPP(B)-pCR2.1. The ligation reaction wastransformed into DH5alpha competent cells (BRL), plated onto LB agarcontaining Ampicillin and incubated overnight at 37° C. Individualcolonies were inoculated into 4 ml LB media containing Ampicillin,incubated overnight at 37° C., and plasmid DNA was isolated using WIZARDDNA Purification System (PROMEGA). The sequence of the PCR fragmentwithin SPP(A)-pCR2.1 and SPP(B)-pCR2.1 included sequences identical toESTs AA375349, AA376229 and AA133909 and contained the putative stopcodon for the SPP phosphatase.

EXAMPLE 3

Construction of hSPP1 Clones for Expression

An 830 base pair BamHI-EcoRI fragment encoding the 3′ end of theputative SPP phosphatase was isolated from the SPP(B)-pCR2.1. A 1222base pair SalI-BamHI fragment encoding the 5′ end of the SPP phosphatasewas isolated directly from gsc:00005190 DNA (amyg2_p0b09 in pBluescriptII SK-). The two restriction fragments were purified with GENECLEAN andligated into E.Coli cloning vector pGEM-3Zf(+) at its SalI and EcoRIsites to give the complete SPP phosphatase coding sequence(SPP-pGEM3). A2.3 Kb HincII-EcoRI fragment containing the complete coding region wasisolated from SPP-pGEM3 and cloned into the mammalian expression vectorpcDNA3.1zeo(−) (INVITROGEN) at its EcoRV and EcoRI sites to make plasmidhSPP1-pcDNA3.1. Ligation reactions were transformed into DH5alphacompetent cells (BRL), plated onto LB agar containing Ampicillin andincubated overnight at 37° C. Individual colonies were inoculated into 4ml LB media containing Ampicillin, incubated overnight at 37° C., andplasmid DNA was isolated using WIZARD DNA Purification System (PROMEGA).The nucleotide sequences of the intact hSPP1 was determined by automatedsequencing.

EXAMPLE 4

Expression of Phosphatase Activity in Mammalian Cells

Transfection-quality DNA was prepared for plasmid hSPP1-pcDNA3.1 usingendotoxin-free QIAGEN Maxi protocol (QIAGEN, Chatsworth, Calif.). Humanembryonic kidney (HEK293) cells were maintained in high glucoseDulbecco's modified Eagle's medium (DMEM) containing 100 U/mlpenicillin, 100 μg/ml streptomycin and 2 mM L-glutamine supplementedwith 10% fetal bovine serum. Cells (1×10₆) were transfected with 20 μgof plasmid DNA by the CaCl₂ procedure using a kit from SPECIALTY MEDIA(Lavallette, N.J.). Cells were harvested 48 hours after transfectionwith enzyme-free dissociation solution (SPECLALTY MEDIA, Lavallette,N.J.). The cells were washed 3 times in cold PBS and then lysed inhypotonic buffer consisting of 1 mM TrisCl pH 7.2 and a proteaseinhibitor cocktail for 10 min at 4° C. Cell debris was removed bycentrifugation at 1,000× g for 5 min at 4° C., and the supernatant fluidwas recentrifuged at 40,000× g for 30 min. The pellet was suspended at aprotein concentration of approximately 2 mg/ml in 40 mM Tris/Cl pH 7.5,protease inhibitor cocktail, and 20% glycerol.Dihydrosphingosine-1-phosphate phosphohydrolase activity was measured in200□1 containing 50 mM KPO₄ pH 7.2, 0.02% tergitol (NP-40), 0.076% BSA,2□M [³H]dihydrosphingosine-1-phosphate (40,000 cpm), 2 mM semicarbazide,and 0.3 to 5 μg of membrane protein. Following a 40 min incubation at37° C., the assay was terminated with 200 μl 7 M NH4OH. One ml ofchloroform:methanol (3:2) was added and 50 μl of the chloroform layerwas counted by liquid scintillation.

Expression of hSPP1 in HEK293 resulted in a 3 to 5 fold increase indihydrosphingosine-1-phosphate phosphohydrolase activity compared tovector transfected cells.

EXAMPLE 5

mRNA Expression of mSPP1 in Mammalian Tissues

Human brain, cancer and multiple tissue Poly(A)+RNA blots (CLONTECH)containing 2 μg of RNA per lane were probed with a 550 bp EcoRI fragmentfrom SPP(A)-pCR2.1 that was gel purified and labeled with ³²P-dCTP byrandom priming. Blots were hybridized in EXPRESSHYB Solution (CLONTECH)at 68° C. for 1 h and washed following the manufacturer's protocol.Bands were quantified using a MOLECULAR DYNAMICS STORM 860 andnormalized to the amount of actin message present.

A single 3.8 kb transcript was detected in 22 of 23 human tissues thatwere surveyed indicating that this gene is ubiquitously expressed inhumans.

EXAMPLE 6

Inhibitors and Activators of SPP Phosphatase Activity

Inhibitors and activators of hSPP1 can be identified in the³H-dihydrosphingosine-1-phosphate phosphatase assay. Compounds dilutedin DMSO, methanol, or other solvent, are added to assays with membranesprepared from cells expressing hSPP1, and dihydrosphingosine-1-phosphatephosphatase activity is measured as in Example 4. Compounds that reducethe ³H-dihydrosphingosine recovered in the chloroform layer areinhibitors of phosphatase activity, while compounds that increase³H-dihydrosphingosine are activators. A semi-high throughputdihydrosphingosine-1-phosphate phosphohydrolase assay can be run in tubestrips with an assay volume of 100 μl containing 50 mM KPO⁴ pH 7.2,0.02% tergitol (NP-40), 2 μM [³H]dihydrosphingosine-1-phosphate (40,000cpm), 2 mM semicarbazide, and membrane protein prepared from hSPP1expressing cells. Following a 45 min incubation at 37° C., the assay canbe terminated with 100 μl 7 M NH₄OH and 0.5 ml of chloroform:methanol(3:2). Using a robotic pipetting station, 50 μl of the chloroform layercan be distributed into a 96-well T-tray (WALLAC). The T-trays can beair dried, scintillant added, and then counted in a Betaplatescintillation counter (WALLAC).

EXAMPLE 7

Yeast Based Screen for Inhibitors of SPP Phosphatase

Yeast sphingoid base phosphate phosphatase activity is not essentialexcept in combination with other mutations in sphingolipid metabolism.One such lethal combination is lbp1□dpl1□sur2□, which will be dependenton functional expression of hSPP1 for growth and survival. To constructthe strain that carries disruptions of the essential combination of 3yeast genes and expresses hSPP1 for growth, hSPP1 can be subcloned intoa yeast expression vector (pRS414-ADH/TRP) and then transformed into adiploid strain lbp1□::LEU2/LBP1, dpl1□::HIS3/dpl1□::HIS3,sur2□::URA3/sur2□::URA3, sporulated, and Trp+, Leu+, His+, Ura+segregants can be isolated. Inhibitors of hSPP1 phosphatase activity areexpected to inhibit the growth of the strain, which can be measured in a96-well or 384-well spectrophotometric assay.

Compounds diluted in DMSO, methanol, or other solvent, are added towells and inoculated with logarithmic phase cells incubated in SC-TRPmedia. After 24 hours incubation at 30° C., the OD₆₀₀ can be measured ina microplate spectrophotometer. Compounds that reduce the OD₆₀₀ comparedto solvent treated cells are potential inhibitors. To distinguishspecific hSPP1 phosphatase inhibitors from compounds that inhibit yeastgrowth via other mechanisms, the compounds can be tested for growthinhibition against a wild-type strain, which does not require SPPphosphatase activity for growth, and the compounds can also be screenedin an in vitro hSPP1 phosphatase assay as described above.

The Examples have been provided as guidance in practicing the inventionand are not limiting of the scope of the invention which is defined bythe following claims.

1. A method of determining whether a candidate compound is an inhibitorof a human sphingosine-1-phosphate phosphatase comprising: (a) providinghost cells harboring an expression vector that includes a polynucleotideselected from the group consisting of: (i) a polynucleotide encoding apolypeptide having the amino acid sequence of SEQ ID NO: 2, and (ii) apolynucleotide having the nucleotide sequence of SEQ ID NO: 1, (b)contacting said cells with the candidate compound to permit theinteraction of the candidate compound with the humansphingosine-1-phosphate phosphatase polypeptide, and (c) determiningwhether the candidate compound is an inhibitor of said humansphingosine-1-phosphate phosphatase polypeptide by ascertaining theactivity of the human sphingosine-1-phosphate phosphatase polypeptide inthe presence of the candidate compound and comparing said activity to ameasurement of the human sphingosine-1-phosphate phosphatase polypeptideactivity of the cells before step (b).
 2. A method of determiningwhether a candidate compound is an inhibitor of a human sphingosine-1-phosphate phosphatase polypeptide comprising: (a) providing a samplethat includes a human sphingosine-1-phosphate phosphatase polypeptidehaving the amino acid sequence of SEQ ID NO: 2, (b) contacting saidsample with the candidate compound to permit the interaction of thecandidate compound with the human sphingosine-1-phosphate phosphatasepolypeptide, and (c) determining whether the candidate compound is aninhibitor of said human sphingosine-1-phosphate phosphatase polypeptideby ascertaining the activity of the human sphingosine-1-phosphatephosphatase polypeptide in the presence of the candidate compound andcomparing said activity to a measurement of the humansphingosine-1-phosphate phosphatase polypeptide activity of the cellsbefore step (b).